Shank is a dose-dependent regulator of Cav1 calcium current and CREB target expression
- Massachusetts General Hospital, United States
- Harvard Medical School, United States
Figures
Figure 1 with 1 supplement
SHN-1 promotes EGL-19/Cav1 channel function.
(A) The protein domains found in SHN-1 and rat Shank3A are compared. SHN-1 lacks an SH3 domain but contains all other domains found in mammalian Shank proteins. Homology between the worm and mammalian protein is shown for each domain. (B–F) Voltage-activated Ca+2 currents were recorded from adult body wall muscles of the indicated genotypes at holding potentials of −60 to +40 mV. Averaged traces (B), mean current density as a function of holding potential (C), normalized conductance as a function of holding potential (D), mean current density at 10 mV (E), and mean deactivation time constants (F) are shown. shn-1 mutants had significantly decreased Ca+2 current-density and this defect was rescued by a single copy transgene expressing SHN-1 in body muscles (nuSi26) (D). No significant differences were observed for voltage-dependence of current activation and de-activation kinetics. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (***p<0.001). Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for this figure are shown in Supplementary file 1.
Figure 1—figure supplement 1
Supplemental data related to Figure 1.
(A–B) The SHN-1 PDZ domain binds to the EGL-19/Cav1 c-terminus in yeast two-hybrid (A) and GST-pull down (B) assays. (C) Potassium current density and voltage-dependence of activation were unaltered in shn-1 mutants. Voltage-activated K+ currents were recorded from adult body wall muscles at holding potentials of −60 to +40 mV. Averaged traces, mean current density (pA/pF) as a function of holding potential, and normalized conductance (G/Gmax) as a function of holding potential are shown. No significant differences were observed. (D) Representation of protein domains found in SHN-1 and the predicted protein products made in the shn-1(tm488) and shn-1(ok1241) mutants. Numbers report amino acid residues for each domain and extent of deletions in the mutants. Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for this figure are shown in Supplementary file 1.
Figure 2 with 1 supplement
SHN-1 binding to EGL-19’s carboxy-terminus promotes the expression or function of L-type calcium channels.
(A) Predicted c-terminal sequences of mutant EGL-19 proteins are shown. egl-19(nu496) is a 22 bp deletion and egl-19(nu495) is a 5 bp deletion, both resulting in frame shifts that delete the carboxy-terminal PDZ ligand of EGL-19 (-VTTLCOOH). Residues in blue represent the PDZ ligand. Residues in red represent those introduced by the frame shift mutations. (B–E) Voltage-activated Ca+2 currents were recorded from adult body wall muscles of the indicated genotypes at holding potentials of −60 to +40 mV. Representative traces (B), mean current density at 0 mV (C, E), and mean current density as a function of holding potential (D) are shown. The egl-19(nu496) and shn-1(tm488) single mutants had similar decreases in Ca+2 current-density, and additive defects were not observed in the double mutant. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (***p<0.001). Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for this figure are shown in Supplementary file 1.
Figure 2—figure supplement 1
Supplemental data related to Figure 2.
Deleting the EGL-19 c-terminal PDZ ligand (in nu496 mutants) had no effect on the kinetics of current deactivation (A) nor on the voltage dependence of current activation (B). Summary data are plotted for recordings shown in Figure 2. (C–F) The shn-1(ok1241) mutation, which deletes the PDZ and part of the proline rich domains (Figure 1—figure supplement 1D), decreases calcium current density (C–D) but has no effect on the voltage-dependence of current activation (E) nor on the kinetics of current deactivation (F). Values that differ significantly from wild type controls are indicated (**p<0.01, *p<0.05). Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for this figure are shown in Supplementary file 1.
Figure 3
SHN-1 promotes EGL-19/Cav1 delivery to the cell surface.
(A–B) Voltage-activating gating currents were significantly decreased in shn-1 null mutants. Averaged trace of gating current in wild type adult body muscles (A) and mean gating charge (normalized to capacitance) (B) are shown. (C–G) Surface delivery of the Terrier fusion protein is significantly reduced in shn-1 null mutants. (C) A schematic illustrating the structure of the Terrier fusion protein is shown. (D) A schematic illustrating the imaged region (left) and representative images of Terrier pHluorin fluorescence in the nerve ring are shown. Mean pHluorin puncta intensity (E), pHluorin puncta area (F), and total pHluorin puncta fluorescence (G) are shown. Regions of interest utilized to quantify Terrier fluorescence are indicated (D). The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (**p<0.01, *p<0.05). Error bars indicate SEM.
Figure 4
Analysis of mRNA abundance following muscle depolarization.
mRNA abundance in Lev (200 µM, 1 hr) versus mock treated synchronized L4 larvae is plotted. Fold change (x-axis) is plotted against the statistical significance (y-axis) for each probeset. Fold changes are shown in log2 scale. Adjusted P values are shown in - log10 scale. Genes with increased (red dots) and decreased (green dots) expression are indicated (>2 Fold-change, FDR p<0.05). Probe sets corresponding to gem-4 and cex-1 are indicated. All genes that are differentially expressed following Lev treatment are listed in Supplementary file 2.
Figure 5 with 1 supplement
gem-4 Copine expression in body muscle is induced by depolarization.
Induction of gem-4 expression was analyzed by qPCR (A) and using a transcriptional reporter (B–H). (A) The abundance of gem-4 mRNA (assessed by qPCR) was increased following 1 hr levamisole (Lev) exposure. The number of biological replicates is indicated. (B) A schematic diagram of the gem-4 reporter construct (left) and representative images of muscle nuclei (right) before and after a 20 min Lev exposure, and 2 hr recovery. (C–D) The mean fold induction of the gem-4 reporter (Pgem-4) after Lev treatment is shown. Lev-induced gem-4 expression was abolished in mutants lacking UNC-29, an essential subunit of the Lev receptor (C) and in mutants lacking the transcription factor CRH-1 (D). The crh-1 mutant defect in gem-4 induction was rescued by a transgene expressing CRH-1 in body muscles (D). (E) Expression of the gem-4 reporter was measured following photo-stimulation of transgenic animals that express ChR2 in cholinergic motorneurons. Expression of gem-4 was significantly increased by 2, 5, 10, and 20 Hz photo-stimulation (for 20 min). Photo-evoked gem-4 expression was not observed when animals were not cultured with ATR. (F–G) The fold induction of the gem-4 reporter following Lev exposure was significantly reduced in shn-1 null mutants (F) but not in shn-1(ok1241) mutants, which lack the PDZ domain (G). Lev-induced gem-4 expression was significantly increased in egl-19(nu496) mutants, which lack the carboxy-terminal PDZ ligand (H). (I–J) Lev induction of the cex-1 reporter is significantly reduced in shn-1 mutants. (I) Schematics of the cex-1 and myo-3 reporters are shown. (J) Expression of the cex-1 reporter (normalized to myo-3 expression in the same nucleus) was significantly increased by Lev treatment. The Lev-induced expression of the cex-1 reporter was significantly reduced in shn-1 mutants. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (***p<0.001; **p<0.01; *p<0.05). Error bars indicate SEM.
Figure 5—figure supplement 1
Supplemental data related to Figure 5.
(A) myo-3 expression in body muscles was unaltered by Lev treatment. Pmyo-3::NLS-mCherry expression (using nuIs525) with and without Lev exposure is plotted for nine genotypes: crh-1, shn-1, 4 copies shn-1, shn-1/+, egl-19(nu496), unc-29, shn-1(ok1241), shn-1(tm488); egl-19(nu496), and unc-13. Equal expression +/- Lev is indicated by the dashed line. (B–C) A mutation that blocks synaptic transmission (unc-13) blocked photo-induced (B) but not Lev-induced (C) gem-4 expression. Error bars indicate SEM.
Figure 6
shn-1 gene dosage regulates calcium current density and gem-4 induction.
The effect of varying shn-1 gene dosage on calcium current density (A–B) and gem-4 induction (C) was analyzed. The following genotypes were analyzed: 0 copies of shn-1 [shn-1(tm488) homozygotes], 1 copy of shn-1 [shn-1(tm488)/+ heterozygotes], 2 copies of shn-1 (wild-type) and 4 copies of shn-1 (nuSi26 homozygotes in wild-type). (A–B) Muscle Ca+2 current was sensitive to changes in shn-1 gene dose, with decreased (0 shn-1 copies) and increased (1 and 4 shn-1 copies) current density observed in the indicated genotypes. Mean current density as a function of holding potential (A), mean current density at 10 mV (B), and mean current deactivation time constants (C) are shown. (D) Lev-induced gem-4 expression was significantly reduced in animals with 0, 1, and 4 copies of shn-1. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (***p<0.001; **p<0.01). Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for panels A-C are shown in Supplementary file 1.
Figure 7 with 1 supplement
Synaptic transmission at the NMJ is not sensitive to shn-1 gene dosage.
Stimulus-evoked EPSCs were recorded from adult body wall muscles. Representative traces of evoked responses (A), averaged shn-1 and WT evoked responses (normalized to equal amplitudes) (B), and mean evoked charge transfer (C) are shown. Evoked charge was significantly increased in shn-1 homozygotes and this defect was rescued by a single copy transgene (nuSi26) that restored SHN-1 expression in body muscles (C). Averaged peak normalized shn-1 and WT evoked responses have indistinguishable rise and decay times, indicating that rise and decay kinetics were unaltered (B). Evoked charge transfer (D) and peak amplitudes (E) did not differ significantly in animals containing 1, 2, and 4 shn-1 copies. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (**p<0.01; ns, not significant). Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for this figure are shown in Supplementary file 1.
Figure 7—figure supplement 1
shn-1 mutations and gene dosage had no effect on mEPSCs.
mEPSCs were recorded from adult body muscles of the indicated genotypes. Representative traces (A), mean amplitude (B,D), and mean rate (C,E) are shown. No significant differences were observed. The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for this figure are shown in Supplementary file 1.
Additional files
-
Supplementary file 1
This table lists means, errors, and p values for all electrophysiology figures.
- https://doi.org/10.7554/eLife.18931.013
-
Supplementary file 2
This table lists Affymetrix probe sets that are differentially expressed following levamisole treatment (>2 fold change, FDR p<0.05).
- https://doi.org/10.7554/eLife.18931.014
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Shank is a dose-dependent regulator of Cav1 calcium current and CREB target expression
eLife 6:e18931.
https://doi.org/10.7554/eLife.18931
